It is now well known that the binding of p53 to its DNA response elements is strongly correlated with its biological function, and many tumorigenic mutants lead to loss of both DNA binding and tumor suppressor function. A recent cocrystal structure of a minimal core p53 DNA binding domain peptide (p53DBD) complexed with a DNA response element has provided valuable insights into the binding specificity of p53. However, many important questions remained unanswered. We have carried out extensive chemical footprinting, cross-linking and DNA cyclization experiments on the complex between wild-type p53DBD and the Waf1 response element. Waf1 is a response element of considerable interest since its gene product, the p21 protein, inhibits CDK function, leading to cell cycle arrest at the G1/S phase boundary. The results show that four p53DBD bind to the major groove of the 20 base pair Waf1 response element, with each peptide bound to a pentameric quarter-site in a staggered array such that the peptides are oriented in the same direction. Molecular modeling studies using crystallographic coordinates for the p53DBD peptide and the protein-DNA contacts recently identified show that severe steric clashes occur when the peptides are bound to straight DNA, but are effectively relieved when the response element DNA bend approximately 50 degrees. This result has not been previously observed in regulatory nucleoprotein complexes and has important implications for the sequence specificity of p53 binding. An important component of the DNA binding properties of p53 is the tetramerization of the active protein. A complete structure of the core tetramerization domain of wild-type p53 has been obtained. This domain has a novel, antiparallel structure that appears to permit interactions of the basic C-terminal tail with the p53DBD, thereby modulating the DNA sequence specificity and binding affinity of wild-type p53. The p53 tetramerization domain is located in the C-terminal region of the protein. This region is phosphorylated at several serines, suggesting that phosphorylation may be an important regulator of p53 function. To determine if phosphorylation affects tetramer formation, we synthesized phosphopeptides corresponding to the C-terminal region of p53 and found that phosphorylation at Ser392 increased the association constant for tetramer formation nearly 10-fold. These results suggest that phosphorylation at Ser392 may modulate the affinity of tetramers, most probably between the C-terminal residues and the region immediately adjacent to Ser315.